This project is concerned with the mechanisms underlying electrical activity-dependent regulation of genes in electrically excitable cells. Electrical activity is known to have a pervasive influence on the type and amount of protein synthesized by both nerve and muscle cells and thus has an important influence on both performance of the adult nervous system and its differentiation during development. Moreover, the ability of the nervous system to regulate gene expression by using signals generated by electrical is likely to have important consequences for the nervous system to be modified with use. This project is designed to identify critical regulatory elements in the genes encoding the skeletal muscle acetylcholine receptor (AChR) and to identify proteins that interact with these regulatory elements. Thus, can we begin to identify and characterize the cellular pathway that couples electrical activity to gene expression. We have isolated and characterized the gene encoding the delta subunit of the murine skeletal muscle AChR and used transient and stable transfection assays to identify a 54 bp cis-acting regulatory element which behaves as an enhancer that controls expression of the delta subunit gene in a cell type-specific and differentiation-dependent manner. Preliminary data indicates that an activity present in nuclear extracts from myotubes binds to this cell type specific enhancer. This project is designed to extend these studies to further characterize control of delta subunit gene expression during development and to use similar technology to characterize regulation of the delta subunit gene by electrical activity. Since a change in gene expression that results as a consequence of altered patterns of electrical activity is a possible mechanism for use-dependent changes in the structure and function of the nervous system, it is important that the steps and mechanisms that couple patterns of electrical activity to control of gene expression are understood. The experiments described in this proposal are likely to provide a basis for a molecular understanding of how genes are regulated in electrical excitable cells. Grant=RO1NS27968 Acetylcholine is well established as a neurotransmitter in the mammalian CNS, mediating a wide variety of responses mainly via activation of muscarinic receptors. The muscarinic system is functionally very important since, in addition to regulating normal neuronal excitability, it is implicated in a number of disease states, including Alzheimer's, Parkinson's and epilepsy, and modulation of long-term potentiation, a candidate mechanism of memory and learning. Recent pharmacological and molecular biological work has revealed a diversity of muscarinic receptor subtypes and associated second messenger systems. The central hypothesis of this proposal is that different muscarinic receptor subtypes mediate different responses. The specific aims of this proposal are to: 1) determine if the subtypes in hippocampus correspond to established subtypes as defined by pharmacological techniques, 2) identify the functional roles of the various subtypes, and 3) investigate the possible involvement of second messengers in muscarinic responses associated with specific subtypes. Several electrophysiological responses to muscarinic agonists will be studied using intracellular techniques (current-clamp and single-electrode voltage-clamp) in the rat hippocampal slice, including: Muscarinic depolarization, block of several ionic conductances and inhibition of G- protein-related transmitter responses. The heterogeneity of muscarinic receptors will be determined quantitatively by Schild plot analysis of the effects of competitive antagonists. The role of second messenger systems in muscarinic responses will be investigated by a pharmacological approach to mimic, inhibit or augment the actions of the systems activated by muscarinic agonists. Deficits in cholinergic systems, including the possible selective loss of particular receptor subtypes, occurs in Alzheimer's and Parkinson's diseases. A better understanding of the function of the muscarinic receptor subtypes could contribute to the rational design of drug therapy for several disease states.